1. Field of the Invention
The present invention relates to a composition of redox-reagent for an electrochemical biosensor including metal-containing complex and thionine or derivative thereof as an electron transfer mediator, and a biosensor comprising the same.
2. Description of the Related Art
While a lot of efforts for improving the accuracy of a glucose meter for managing diabetes have been consistently made for the last thirty years, there are mainly two glucose measuring methods, i.e., spectrophotometer measurement and electrochemical measurement.
Between these two, the electrochemical measurement is more common, because its accuracy deterioration caused by blood-contamination onto a glucose meter is relatively less frequent and it requires smaller amount of blood.
Despite many researches associated with a glucose meter for such a long period of time, many problems still remain unsolved with regard to the accuracy of the glucose meter, and one of the problems is inherent to enzymes that are employed for the measurement.
For example, FAD-GOx, which is the most commonly used glucose redox enzyme for commercial electrochemical sensors, is stable under heat and has excellent specificity in enzymatic reaction, however, the measured glucose value is affected by the oxygen concentration in blood.
While a sensor based on PQQ-GDH enzyme is free from the oxygen influence, many monosaccharides and disaccharides including mannose, maltose, or lactose still affect the sensor (Igarashi, S., and Sode, K., Engineering PQQ glucose dehydrogenase with improved plate specificity-first site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase. Biomol. Engineer., 21, 81-89 (2004)), and more specifically, diabetes community has been keen on the effect of high maltose concentration found in diabetics (Mehmet, S., Quan, G., Thomas, S., and Goldsmith, D., Important causes of hypoglycemia in patients with diabetes on peritoneal dialysis. Diabet. Med., 18, 679-682 (2001)).
A NAD-GDH enzyme has excellent specificity on glucose reaction, however, its usage is inconvenient because it requires NAD+ or NADP+ be added to the reagent composition. As a solution to this inconvenience, an electrochemical sensor based on FAD-GDH (Enzyme Commission No. 1.1.99.10) has been developed.
The sensor based on FAD-GDH enzyme is not affected by the amount of oxygen in blood. Therefore, the sensor can be effectively applied to any blood sample collected from veins, arteries, or capillaries in human body. Although the sensor has a downside of reacting with xylose, it does not react with mannose, maltose or lactose, thus leaving it relatively superior to (PQQ-GDH)-based sensor with regard to glucose specific reaction.
The accuracy of a sensor is affected by not only the enzymes but also the electron transfer mediator. Potassium ferricyanide ([K3Fe(CN)6]), phenazine-methosulfate, methoxy phenazine-methosulfate, phenazine methyl sulfate and dichloroindophenol are well known for FAD-GDH's electron transfer mediator. However, the properties of all the above-mentioned mediators easily change at high temperature or humidity condition, leading to a tendency of accuracy deterioration in sensor when the sensor is stored for a long period of time. To address this problem, a method of replacing potassium ferricyanide with hydrophilic derivatives of phenothiazine was suggested (US 20090145775).
For an electrochemical sensor based on FAD-GOx (EC No. 1.1.3.4), hexaamineruthenium chloride ([Ru(NH3)6Cl3]) has been employed as an electron transfer mediator because the sensor is much less affected from interfering substances such as uric acid and gentisic acid, and because its accuracy is much less deteriorated from moisture compared to a sensor employing ferricyanide (U.S. Pat. No. 7,288,174, US20090280551). However, because of fairly slow reaction between hexaamineruthenium chloride and FAD-GDH (EC No. 1.1.99.10), fabricating a useful sensor has not been easy
Meanwhile, the following is a list of the examples—using two types of electron transfer mediators.
EP 0238322 A1 teaches a method of increasing the speed of electron transfer between bacteria and an electrode with ferricyanide and benzoquinone.
US 20070295616 A1 teaches a method of applying osmium (Os) and ferricyanide to a glucose sensor.
A. Amine et al. (A. Amine, J. M. Kauffmann, G. J. Patriarche, G. D. Christian; Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrode, Talanta, 1993, 40, 1157-1162.) teach a method of applying phenazine methosulfate and ferricyanide to glucose oxidase.
A. Amid and J. M Kauffman (A. Amine and J. M. Kauffmann; Preparation and characterization of a fragile enzyme immobilized carbon paste electrode, Bioelectrochem. Bioenerg., 1992, 28, 117-125.) teach a method of applying phenazine methosulfate and ferricyanide to glutamate dehydrogenase.
Yet, a secondary electron transfer mediator which can be used along with ruthenium complex has not been found.
In a research to find a solution to the problems mentioned above, the present inventors found that a reagent composition including metal-containing complex and thionine or derivatives thereof as an electron transfer mediator improves the efficiency of glucose detection by increasing the reaction speed between redox enzyme-thionine (or derivative thereof) and a metal-containing complex and that the reagent composition is hardly affected under high humidity condition and from interfering substances, and thus, completed the present invention.